SUMMARY (CORE B) Research in this P01 will depend on unique antibody reagents that are developed and implemented by the Immunology Core B. Monoclonal antibodies (MAbs) generated by Core B from memory B cells (MBCs) will be essential to address critical questions about the epitopes and lineage of neutralizing antibodies produced after natural dengue virus (DENV) infection or immunization with a tetravalent live attenuated dengue vaccine. Core B will meet these needs and provide infrastructure support for Projects 1 and 2 of the P01. Specifically, the Core B facility will receive frozen PBMCs from individuals who have experienced documented repeat DENV infections from Nicaragua, as has previously been done to successfully isolate human MAbs. Core B also will receive PBMCs from DENV-nave and DENV-exposed recipients of the DENVax tetravalent vaccine candidate from Phase 2 clinical trials. MAbs will be generated from MBCs after EBV transformation or DENV-specific MBC sorting and identification of wells containing DENV-specific antibodies, followed by electrofusion with a myeloma partner cell to create stable hybridomas or recombinant cloning and expression of heavy and chain variable regions. During this process, Core B will produce recombinant DENV proteins and purified viruses for limited initial characerization that will be performed by the Core (e.g., single dilution neutralization, type- specificity vs cross-reactivity, E vs prM protein specificity) to facilitate prioritization of clones for fusion and large-scale MAb production. Purified MAbs then will be sent to the laboratories of Project 1 (natural infections) and Project 2 (vaccinees) for more detailed characterization of type specificity, epitope mapping, and functional anaysis. Core B will also provide Projects 1 and 2 with recombinant DENV proteins from Nicaraguan or vaccine strains, respectively. Based on characterization by Projects 1 and 2, the Core will sequence the antibody genes of all MAbs of functional interest and then perform high throughput sequence analysis of total B cell repertoires from PBMCs collected from prior natural infections in the same individuals (Project 1) or PBMCs collected prior to vaccination in DENV-exposed recipients (Project 2) in order to identify ?siblings? of neutralizing antibodies isolated at later time-points. We hypothesize that highly neutralizing cross-reactive MAbs isolated at later time-points are derived from pre-existing MBCs from a prior DENV exposure that have undergone further somatic hypermutation and affinity maturation. Overall, Core B will provide platforms for generating human MAbs from naturally-infected or vaccinated individuals and sequencing antibody lineages. The functional analysis of these antibody repertoires by Projects 1 and 2 will help to elucidate correlates of humoral immune protection after natural infection and immunization.